The present invention relates to a new Type II restriction endonuclease, PacI, obtainable from Pseudomonas alcaligenes, and to process for producing the same.
Many bacteria contain systems which guard against invasion of foreign DNA. Bacterial cells contain specific endonucleases that make double-strand scissions in invading DNA unless the DNA has been previously modified, usually by the appropriate corresponding DNA methylase. The endonuclease with its accompanying methylase is called a restriction-modification system (hereinafter "R-M system"). The principle function of R-M systems is thus defensive: they enable bacterial cells to resist infections by bacteriophage and plasmid DNA molecules which might otherwise parasitize them.
Bacteria usually possess only a small number of restriction endonucleases per species. The endonucleases are named according to the bacteria from which they are derived. Thus, the species Haemophilus aegyptius, for example synthesizes three different restriction endonucleases, named HaeI, HaeII, and HaeIII. These enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively. Escherichia coli RY13, on the other hand, synthesizes only one restriction endonuclease, Eco RI, which recognizes the sequence GAATTC.
Restriction endonucleases, the first component of R-M systems, have been characterized primarily with respect to their recognition sequence and cleavage specificity because of their practical use for molecular dissection of DNA. The majority of restriction endonucleases recognize sequences 4-6 nucleotides in length. More recently, recognition endonucleases having recognition sequences of 7-8 nucleotides in length have been found. Most, but not all, recognition sites contain a dyad axis of symmetry, and in most cases, all the bases within the site are uniquely specified. This symmetrical relationship in the recognition sequence of restriction endonucleases has been termed "palindromes". Some restriction endonucleases have degenerate or relaxed specificities in that they can recognize multiple bases at the same positions. HaeIII, which recognizes the sequence GGCC is an example of restriction endonuclease having a symmetrical relationship, while HaeII, which recognizes the sequence PuGCGCPy, typifies restriction endonucleases having a degenerate or relaxed specificity. Endonucleases with symmetrical recognition sites generally cleave symmetrically within or adjacent to the recognition site, while those that recognize asymmetric sites tend to cut at distance from the recognition site, typically from about 1-18 base pairs away from the site.
The second component of bacterial R-M systems are the modification methylases. These enzymes are complementary to restriction endonucleases and provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following methylation, the recognition sequence is no longer bound or cleaved by the corresponding restriction endonuclease. The DNA of a bacterial cell is always fully modified, by virtue of the activity of its modification methylase and it is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack.
More than 1000 different restriction endonucleases have been isolated from bacterial strains, and many share common specificities. Restriction endonucleases which recognize identical sequences are called "isoschizomers". Although the recognition sequences of isoschizomers are the same, they may vary with respect to site of cleavage (e.g., XmaI V. SmaI Endow, et al., J. Mol. Biol. 112:521 (1977); Waalwijk, et al., Nucleic Acids Res. 5:3231 (1978)) and in cleavage rate at various sites (XhoI v. Pae R7I Gingeras, et al., Proc. Natl. Acad. Sci U.S.A. 80:402 (1983)).
Specific Type II restriction endonucleases are already known for numerous DNA sequences, however, a large number of restriction enzymes with diversified enzymatic characteristics are necessary for successful genetic manipulation. In particular, restriction endonucleases that require eight specific nucleotides in their recognition sequence are quite rare. In fact, to date only three have been identified, NotI (GCGGCCGC), SfiI (GGCCNNNNNGGCC), and FseI (GGCCGGCC).
Accordingly, there is a continued need for Type II restriction endonucleases which recognize novel DNA sequences, and in particular, those which recognize eight nucleotides recognition sequences.